Disseminated tumor cells can enter into a dormant state for months to years, evading the immune system and chemotherapies that largely target actively proliferating cells. Tumor dormancy is one of the main mechanisms resulting in tumor relapse. Even if a patient goes into remission after surgery and/or chemotherapy, they can relapse months or even years later when the dormant tumor cells begin proliferating again. How these dormant tumor cells make the decision to begin proliferating again and why they are typically more aggressive than the original primary tumor remains unknown. We aim to carry out single-cell time-lapse imaging studies to watch as tumor cells plated in 3D matrix enter a dormant state and use fluorescent biosensors of key cell cycle regulators to understand the molecular events resulting in dormancy. We also aim to follow those same cells for several days and study when and how they begin proliferating again. We have been developing the imaging and analysis methods to be able to track cells in 3D over several days. We are currently investigating how different extracellular matrices impact the ability of cells to enter into and out of a dormant state.